Mitochondrial Cholesterol Side-chain Cleavage Activity Research Articles

Cyclosporin-Mediated Depression of Luteinizing Hormone Receptors and Heme Biosynthesis in Rat Testes: A Possible Mechanism for Decrease in Serum Testosterone*

The toxic side-effects of the immunosuppressive drug cyclosporin (CsA) include testicular dysfunction and a decline in circulating testosterone. However, mechanisms for the consistently observed CsA-mediated depression of serum testosterone levels are unclear because of conflicting reports concerning circulating gonadotropin levels and incomplete studies of intratesticular steroidogenesis. To elucidate these mechanisms, endocrine-regulated testicular steroidogenesis and heme metabolic parameters were studied in male rats given sc injections of either 25 or 40 mg/kg.day CsA for 6 days and then killed on the seventh day. Consistent with earlier reports, CsA treatment dramatically suppressed serum testosterone levels (less than 20% of control at both CsA doses). Additionally, the intratesticular testosterone content declined with the higher CsA dose. Serum LH and FSH levels were elevated up to 2- to 4-fold after the higher CsA treatment regimen. Measurement of decreases in testicular receptors for LH revealed for the first time that CsA treatment significantly reduced the ability of the testes to respond to normal or elevated circulating levels of LH. In animals receiving higher dose of the drug, cytochrome P-450-dependent mitochondrial cholesterol side-chain cleavage activity, which is the rate-limiting step in steroidogenesis, was markedly reduced to a mere 30% of the control value. Additionally, the activity of the microsomal cytochrome P-450-dependent 17 alpha-hydroxylase was decreased to less than half of the control value. Biotransformation of the prototype drug, benzo(a)pyrene, as well as microsomal cytochrome P450 levels declined significantly after the higher CsA dose, suggesting that CsA has an adverse affect on testicular cytochromes P-450 in general. In addition, CsA treatment altered heme metabolic parameters; significant increases in the activity of uroporphyrinogen-I synthetase and total porphyrin content were noted. Conversely, the activity of ferrochelatase, the enzyme that incorporates iron into porphyrin to form heme molecule, decreased significantly, as did the total heme levels. The latter was reduced to only 61% of control values. The findings suggest the likelihood that the observed inhibition of heme formation may contribute substantially to the reduced levels of microsomal cytochromes P-450 and steroidogenic activities that depend on them. Taken collectively, these data suggest a plausible mechanism by which CsA may induce testicular dysfunction; as the result of a combination of reduction in the number of LH receptors and a suppression of heme formation, the hemoprotein-dependent steroidogenic enzymes activities are compromised, leading to an impairment of normal testicular function.

Further characterization of the inhibitory effect of monensin on adrenal steroidogenesis

We have previously reported that treatment of cultured mouse adrenal tumor cells with 0.6-1.2 μ M monensin, a monovalent carboxylic ionophore, results in disruption of the organized structure of the Golgi complex. This is associated with an inhibition of adrenocorticotropic hormone (ACTH) or dibutyryl cAMP-stimulated steroidogenesis and impairment of mitochondrial cholesterol side-chain cleavage activity. The present report describes further investigations regarding possible mechanisms for the inhibition. Monensin inhibits both synthesis of fluorogenic steroids and incorporation of [ 14C]acetate into the end-product steroid 11β,20α-dihydroxy-4-pregnen-3-one. Supplementation of monensin-treated cells with 25-hydroxycholesterol, a readily available substrate for steroidogenesis, does not reverse the inhibitory effect on the reaction. The incorporation of l-[ 35S]methionine into trichloroacetic acid precipitable proteins in the isolated mitochondria of monensin-treated cells is inhibited approximately by 40%, whereas the inhibitory effect on the proteins in the cell homogenate is marginal. These findings suggest that a deficiency of newly synthesized proteins in mitochondria, rather than the availability of the substrate cholesterol, may be the primary factor causing impairment of steroidogenesis.

Cholesterol sulfate is a naturally occurring inhibitor of steroidogenesis in isolated rat adrenal mitochondria

We previously reported (Lambeth, J. D., Xu, X. X., and Glover, M. (1987) J. Biol. Chem. 262, 9181-9188) that exogenously added cholesterol sulfate inhibits the conversion of cholesterol to pregnenolone in isolated adrenal mitochondria, and does so by affecting intramitochondrial cholesterol movement but not its subsequent metabolism to pregnenolone by cytochrome P-450scc. We now report that a major kinetic component of the inhibition is noncompetitive with respect to cholesterol, consistent with an allosteric effect at a site other than the substrate binding site of cytochrome P-450scc. We now also report that cholesterol sulfate is present as an endogenous compound in preparations of adrenal mitochondria. Its content varied from 0.05 to 0.8 nmol/mg protein. Cholesterol sulfate level correlated inversely with the mitochondrial cholesterol side-chain cleavage activity. Endogenous cholesterol sulfate thus appeared to account for the variable rates of pregnenolone synthesis which were seen in different mitochondrial preparations. Cholesterol sulfate was metabolized to pregnenolone sulfate by a mitochondrial side-chain cleavage system, but proved to be a relatively poor substrate for an extramitochondrial steroid sulfatase activity present in adrenal cortex. Confirming a role as a naturally occurring inhibitor, removal of endogenous mitochondrial cholesterol sulfate by metabolism to pregnenolone sulfate correlated with a 3-fold activation of cholesterol side-chain cleavage. We suggest that cholesterol sulfate functions in steroidogenic tissues to regulate the magnitude of the steroidogenic response.

Regulation of progesterone biosynthesis by estrogen during baboon pregnancy: placental mitochondrial cholesterol side-chain cleavage activity in antiestrogen (ethamoxytriphetol, MER-25)-treated baboons.

We determined whether the reduction in placental progesterone (P4) production observed after administration of the antiestrogen ethamoxytriphetol (MER-25) to pregnant baboons was associated with a decline in activity and/or content of the placental mitochondrial cholesterol side-chain cleavage system (P-450scc). Pregnant baboons (Papio anubis) were untreated (n = 9) or administered MER-25 (25 mg/kg BW, orally; n = 6) daily on days 140-170 of gestation (term = 184 days). Placentas were obtained by cesarean section on day 170 of gestation, and P-450scc activity and cytochrome P-450 content were determined on mitochondria-rich fractions. Administration of MER-25 to pregnant baboons resulted in a 40% reduction (P less than 0.01, by Student's t test) in the mean (+/- SE) peripheral serum P4 concentration (6.3 +/- 0.3 ng/ml) compared to that in untreated (10.4 +/- 0.3 ng/ml) baboons. P-450scc activity, as determined by formation of pregnenolone (P5) and P4 during a 30-min incubation (picomoles per mg protein), was 37% lower (P less than 0.01) in placental mitochondria obtained from MER-25-treated baboons (179.8 +/- 25.0) than in that from untreated (285.4 +/- 13.4) baboons. Mitochondrial cytochrome P-450 content, assessed by spectral analysis, was 28% lower (P less than 0.02) in antiestrogen-treated (46.7 +/- 2.1 pmol/mg protein) than in untreated (64.8 +/- 5.2 pmol/mg protein) baboons. The initial (time zero) free cholesterol content (nanomoles per mg protein) of mitochondrial-rich preparations was not significantly different in antiestrogen-treated (189.3 +/- 13.0) and untreated (225.0 +/- 15.1) animals. Collectively, these results suggest that the decline in placental P4 production observed in baboons in response to MER-25 occurs at least in part as a result of a decrease in cytochrome P-450scc activity. The loss in P-450scc activity appears to be an intramitochondrial event and not a result of depletion of the total mitochondrial cholesterol pool. We propose, therefore, that one mechanism by which estrogen may regulate the production of P4 by the placenta during primate pregnancy is via the maintenance of placental mitochondrial cytochrome P-450, the terminal oxidase of cytochrome P-450scc.

Changes in adrenocortical monooxygenase activities in alloxan-diabetic rabbits

Studies were carried out to determine if diabetes mellitus influenced the activities of adrenal steroidogenic enzymes. Adult male rabbits were made diabetic by an i.v. infusion of alloxan (100 mg/kg) and were killed 1 or 2 months later. Mitochondrial cytochrome P-450 concentrations were not affected by diabetes but steroid 11β-hydroxylase activity was greater in the diabetics than in controls after both 1 and 2 months. The type I spectral change produced by 11-deoxycorticosterone, the substrate for 11β-hydroxylation, was also greater in mitochondria from diabetics. By contrast, mitochondrial cholesterol side-chain cleavage activity was similar in controls and diabetics. Microsomal cytochrome P-450 concentrations were unaffected by diabetes but 21-hydroxylase activity was significantly lower in adrenal microsomes from diabetics than from controls. The results indicate that alloxan-induced diabetes alters adrenocortical steroid metabolism which may contribute to changes in the pattern of steroid secretion noted by other investigators.

Clomiphene and tamoxifen inhibit the cholesterol side-chain cleavage enzyme activity in hen granulosa cells.

A radiochemical assay was utilized to study the inhibitory effects of clomiphene and tamoxifen on the cholesterol side-chain cleavage enzyme activity in a mitochondrial preparation of granulosa cells isolated from mature ovarian follicles of laying hens. At saturating substrate concentrations, both clomiphene and tamoxifen were able to suppress enzyme activity in a dose-related manner (IC50 1.8 X 10(-5) M). Double reciprocal plots of kinetic data show that the inhibition is mixed, exhibiting competitive kinetics at low concentrations, whereas at high concentrations, the inhibition is of a non-competitive nature. The competitive inhibition constants as determined from Dixon plots are 2 X 10(-5) M for clomiphene and 2.3 X 10(-5) M for tamoxifen. It is concluded that, in granulosa cells, clomiphene and tamoxifen directly inhibit the mitochondrial cholesterol side-chain cleavage activity. This inhibition may represent an important aspect of the mode of action of clomiphene and tamoxifen.

Restoration effects of exogenous luteinizing hormone on the testicular steroidogenesis and Leydig cell ultrastructure.

In the present study, we explored the restoration effects of exogenous LH on Leydig cell ultrastructure and testicular steroidogenesis in rats that were deprived of endogenous LH via treatment with testosterone-17 beta-estradiol-filled Silastic implants for 10 days. Exogenous LH was supplied continuously via Alzet miniosmotic pumps at the rate of 1 microgram/h for 3, 6, 12, and 24 h or 1, 2, 4, 8, and 12 days. Testes were then perfused in vitro with medium containing 1) LH, 2) 20 alpha-hydroxycholesterol, or 3) pregnenolone substrate, which allowed us to assess LH-stimulated testosterone secretion, cholesterol side-chain cleavage activity, or the conversion of pregnenolone to testosterone, respectively. Other testes were perfusion fixed via the testicular artery for morphometric measurement of Leydig cell number and volume per testis and the surface area of Leydig cell cytoplasmic smooth endoplasmic reticulum (SER), inner mitochondrial membrane, and outer mitochondrial membrane. The results verified that Leydig cell smooth endoplasmic reticulum and inner and outer mitochondrial membrane surface areas are drastically diminished (P less than 0.05 vs. intact controls) by LH withdrawal. Also, the results verified that exogenous LH administered in situ restores Leydig cell ultrastructure and capacity to biosynthesize testosterone. However, the recovery of Leydig cell structure and steroidogenic reactions occurred at strikingly different rates upon restoration of LH after 10 days of the treatment with testosterone-17 beta-estradiol implants. For example, the restoration of testicular capacity to synthesize progesterone in response to LH stimulation or 20 alpha-hydroxycholesterol substrate was completed within 24 h. In contrast, the restoration of Leydig cell SER and testicular capacity to synthesize testosterone from pregnenolone was completed only after 8 days of continuous LH treatment (P greater than 0.05 vs. intact controls). Thus, our results show that LH rapidly restores Leydig cell post-LH receptor steroidogenic events up to and including cholesterol side-chain cleavage activity. Interestingly, there is no temporal association between the recovery of cholesterol side-chain cleavage activity and the surface area of inner mitochondrial membrane surface area. In contrast, 8 days are required to coincidentally restore SER surface area and the capacity of Leydig cells to synthesize testosterone from pregnenolone. We conclude that different cellular mechanisms are involved in the LH-dependent restoration of inner mitochondrial cholesterol side-chain cleavage activity and SER-associated conversion of pregnenolone to testosterone.

Mechanisms subserving the trophic actions of insulin on ovarian cells. In vitro studies using swine granulosa cells.

Direct actions of insulin on gonadal tissues have been difficult to demonstrate in vivo. We have developed an in vitro system in which swine ovarian cells remain highly responsive to trophic actions of insulin. Purified porcine insulin significantly augmented the biosynthesis and secretion of progesterone by cultured granulosa cells. These stimulatory actions of insulin were dose- and time-dependent and saturable. Under serum-restricted conditions, insulin also significantly amplified the capacity of estradiol and 8-bromo cyclic AMP to stimulate progesterone production. Inhibitors of protein and RNA synthesis (cycloheximide, actinomycin D, and alpha-amanatin) inhibited insulin action. The stimulation of progesterone production by insulin was attributable to increased biosynthesis of pregnenolone, rather than diminished catabolism of progesterone to its principal metabolite, 20alpha-hydroxypregn-4-en-3-one. Insulin also enhanced progesterone production in the presence of a soluble sterol substrate, 5-cholesten-3beta,25-diol, which readily gains access to the mitochondrial cholesterol side-chain cleavage system. Moreover, exposure of granulosa cells to insulin produced a three- to sevenfold increase in mitochondrial content of cytochrome P-450 measured by difference spectroscopy, with a corresponding increase in mitochondrial cholesterol side-chain cleavage activity. The capacity of insulin to facilitate progesterone biosynthesis by ovarian cells was mimicked by the insulinlike somatomedin, multiplication stimulating activity, but not by epidermal growth factor, fibroblast growth factor, or porcine relaxin. Insulin's augmentation of progesterone production reflected a selective action on progestin biosynthesis, since insulin significantly suppressed estrogen biosynthesis by granulosa cells.Thus, our investigations indicate that insulin acts on ovarian cells selectively to stimulate pregnenolone (but not estrogen) biosynthesis. The actions of insulin are exerted by processes that require protein and RNA synthesis, and by mechanisms that augment mitochondrial cytochrome P-450 content and facilitate the utilization of cholesterol in the side-chain cleavage reaction. The striking mimicry of insulin effect by multiplication stimulating activity suggests that insulin action may be mediated through somatomedin receptors. Moreover, in view of the high concentrations of somatomedin in ovarian follicles in vivo, our in vitro observations suggest that specific trophic actions of insulin or insulinlike growth factors are likely to significantly regulate the differentiated function of the Graafian follicle in vivo.

The role of phosphoproteins and newly synthesized proteins in the hormonal regulation of steroidogenesis in leydig cells

LH activation of steroidogenesis in Leydig cells is accompanied by increased phosphorylation of at least 5 phosphoproteins. The 17000 Da protein is localized in nuclei and appears not to be involved in the acute stimulation of steroidogenesis. The 57,000 Da protein is the regulatory subunit of the cAMP-dependent protein kinase. The 22,000, 24,000 and 33,000 phosphoproteins are present in microsomes and may regulate the synthesis of specific rapidly-turning-over protein(s) which in turn may stimulate the mitochondrial cholesterol side-chain cleavage activity.

Regulation of cytochrome P-450scc in immature porcine granulosa cells by FSH and estradiol.

FSH, estradiol, or a combination of FSH and estradiol enhanced secretion of progesterone by primary cultures of immature porcine granulosa cells; they also increased the mitochondrial content of cytochrome P-450 and mitochondrial cholesterol side-chain cleavage activity. FSH or estradiol alone increased mitochondrial cytochrome P-450 levels and cholesterol side-chain cleavage activity to a similar extent. The combination of FSH and estradiol further increased cytochrome P-450 and side-chain cleavage activity. Our results demonstrate that FSH, estrogen and the combination of the hormones stimulate progestin synthesis by granulosa cells, at least in part, by increasing cholesterol side-chain cleavage enzyme levels. These observations suggest a dual hormonal regulation of the enzyme catalyzing the rate-limiting step in the biosynthetic pathway of steroid hormones.